Real time electronic cell sensing system and applications for cytoxicity profiling and compound assays

• US 20050213374A1
• Filed: 02/09/2005
• Published: 09/29/2005
• Est. Priority Date: 12/20/2002
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: ACEA Biosciences, Inc (Agilent Technologies, Inc.)
• Inventors: Wang, Xiaobo, Xu, Xiao, Abassi, Yama
• Granted Patent: US 7,560,269 B2
• Time in Patent Office: Days
• US Class Current: 365/156
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: G01N 27/02 by investigating impedance
• CPC Class Codes: G01N 33/4836 using multielectrode arrays
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

The present invention includes devices, systems, and methods for assaying cells using cell-substrate impedance monitoring. In one aspect, the invention provides cell-substrate impedance monitoring devices that comprise electrode arrays on a nonconducting substrate, in which each of the arrays has an approximately uniform electrode resistance across the entire array. In another aspect, the invention provides cell-substrate monitoring systems comprising one or more cell-substrate monitoring devices comprising multiple wells each having an electrode array, an impedance analyzer, a device station that connects arrays of individual wells to the impedance analyzer, and software for controlling the device station and impedance analyzer. In another aspect, the invention provides cellular assays that use impedance monitoring to detect changes in cell behavior or state. The methods can be used to test the effects of compounds on cells, such as in cytotoxicity assays. Methods of cytotoxicity profiling of compounds are also provided.

Claims

1. A device for monitoring cell-substrate impedance, comprising:

a) a nonconducting substrate;

b) two or more electrode arrays fabricated on said substrate, wherein each of said two or more electrode arrays comprises two electrode structures;

c) two or more fluid containers on said substrate, wherein each of said two or more arrays is associated with one of said two or more fluid containers; and

d) at least two connection pads, each of which is located on an edge of said substrate;

wherein for each of said two or more electrode arrays, each of said two electrode structures comprises multiple electrode elements and the first of said two electrode structures of each of said at least two electrode arrays is connected to one of said at least two connection pads, and the second of said two electrode structures of each of said at least two electrode arrays is connected to another of said at least two connection pads;

further wherein at least two of said two or more electrode arrays share one common connection pad;

further wherein each electrode array has an approximately uniform electrode resistance distribution across the entire array; and

further wherein said substrate has a surface suitable for cell attachment or growth;

wherein said cell attachment or growth on said substrate can result in a detectable change in impedance between or among said electrode structures within each electrode array.

2. A system for monitoring cell-substrate impedance, comprising:

a) one or more devices of claim 1, wherein said at least two fluid containers are two or more wells, wherein at least two of said two or more wells of said device comprise an electrode array at the bottom of the well, wherein said electrode array is individually addressed, further wherein said device can be used to measure differences in impedance values that relate to cell behavior;

b) an impedance analyzer;

c) a device station comprising electronic circuitry that can engage said device and selectively connect said two or more electrode arrays of said device to said impedance analyzer; and

d) a software program that can control said device station and record and analyze data obtained from said impedance analyzer.

58. A method of performing an assay to evaluate the effect of one or more test compounds on cells, comprising:

providing the cell-substrate impedance monitoring system of claim 2;

adding cells to two or more wells of said system;

adding different concentrations of a test compound to said two or more of said three or more wells comprising cells to form twor more compound ls comprising different concentrations of said test compound;

providing at least one additional well as a control well to which cells are added that does not receive test compound,

monitoring impedance from said two or more test compound wells and said at least one control well to obtain impedance measurements from said two or more compound wells and said at least one control well at at least one time point before the addition of different concentrations of a test compound to said compound wells and at three or more time points after adding said different concentrations of a test compound to said three or more compound wells;

analyzing said impedance measurements from said two or more compound wells and said at least one control well to evaluate the effect of said test compound on cells.

59. The method of claim 58, wherein said monitoring impedance comprises monitoring impedance at regular or irregular time intervals for an assay period of from minutes to days.

60. The method of claim 58, wherein said analyzing comprises plotting said impedance measurements from said three or more compound wells that comprise different concentrations of a compound versus time to obtain three or more compound concentration impedance curves and plotting said impedance measurements for said at least one control well versus time to obtain at least one control impedance curve.

61. The method of claim 60, further comprising comparing at least one of said three or more compound concentration impedance curves with said control impedance curve to evaluate an effect of said test compound on cells.

62. The method of claim 58, wherein said analyzing comprises deriving a cell index, a normalized cell index, or a delta cell index from said impedance measurements from said three or more compound wells that comprise different concentrations of a compound and from said at least one control well.

63. The method of claim 62, further comprising plotting said cell index, said normalized cell index, or said delta cell index of said at least three compound wells that comprise different concentrations of a compound versus time to provide three or more compound concentration CI curves and plotting said cell index, said normalized cell index, or said delta cell index of said at least one control well versus time to provide at least one control CI curve.

64. The method of claim 63, further comprising comparing at least one of said compound concentration CI with said at least one control CI curve to evaluate the effect of said test compound on cells.

65. The method of claim 64, wherein said assay is an assay for cell viability, cell adhesion, apoptosis, cell differentiation, cell proliferation, cytotoxicity, cell morphology change, cell cycle change, IgE-mediated cell activation or stimulation, receptor-ligand binding, cell number quantification, cell quality control, time-dependent cell cycling monitoring, detection or quantification of neutralizing antibodies, specific T-cell mediated cytotoxic effect, viral or bacterial toxin mediated cell pathologic changes, or cell death;

and

wherein evaluating the effect of said test compound on cells comprises determining a change in cell status in response to at least one of said different concentrations of said test compound, wherein a change in cell status comprises at least one of;

cell attachment or adhesion status, cell growth or proliferation status;

the number of viable cells or dead cells;

cytoskeleton change or re-organization;

or the number of cells going through apoptosis or necrosis.

66. The method of claim 62, further plotting said cell index, said normalized cell index, or said delta change index versus concentration for at least one time point of said assay to generate one or more dose response curves for one or more time points at which impedance is measured.

69. The method of claim 62, further comprising deriving a cell change index or a second order derivative of a cell index from said impedance measurements from at least one of said three or more compound wells that comprise different concentrations of a test compound and from said at least one control well.

70. The method of claim 69, wherein said cell change index or a second order derivative of a cell index is a cell change index (CCI).

71. The method of claim 70, further comprising determining the CCI at a given time point for at least one of said three or more compound wells that comprise different concentrations of a test compound and said at least one control well as being either:

approximately equal to 0.7, much greater than 0.7, greater than zero and less than 0.7, approximately equal to zero, less than zero, or much less than zero;

wherein a CCI approximately equal to 0.7 indicates log rate growth, a CCI much greater than 0.7 indicates faster than log rate growth, a CCI greater than zero and less than 0.7 indicates slower than log rate growth, a CCI approximately equal to zero indicates no growth (a constant cell index), a CCI less than zero indicates cells are detaching from the substrate, and a CCI much less than zero indicates cell are detaching rapidly from the substrate.

72. The method of claim 70,

further comprising plotting said cell change index from said at least one of said three or more compound wells that comprise different concentrations of a compound and from said at least one control well versus time to obtain at least one test compound CCI curve and at least one control CCI curve;

and

further comprising comparing said at least one test compound CCI curve with said at least one control CCI curve.

73. The method of claim 72, wherein evaluating the effect of said test compound on cells comprises determining a change in cell status in response to at least one of said different concentrations of said test compound, wherein a change in cell status comprises at least one of:

cell attachment or adhesion status, cell growth or proliferation status;

the number of viable cells or dead cells;

cytoskeleton change or re-organization;

or the number of cells going through apoptosis or necrosis.

74. The method of claim 73, wherein said assay is an assay for cell viability, cell adhesion, apoptosis, cell differentiation, cell proliferation, cytotoxicity, cell morphology change, cell cycle changes, IgE-mediated cell activation or stimulation, receptor-ligand binding, cell number quantification, cell quality control, time-dependent cell cycling monitoring, detection or quantification of neutralizing antibodies, specific T-cell mediated cytotoxic effect, viral or bacterial toxin mediated cell pathologic changes, or cell death.

3-57. -57. (canceled)

67. The method of claim 166, further comprising using said one or more dose-response curves to calculate a time-dependent IC5, IC10, IC20, IC30, IC40, IC50, IC60, IC70, IC80, IC90, or IC95 at one or more time points for said compound.

68. The method of claim 67, comprising using said one or more dose-response curves to calculate one or more time-dependent IC50 for said compound for one or more time points.

75-84. -84. (canceled)